A new method for normodense eosinophil purification is proposed. It is based on the use of high-density Ficoll-Hypaque (density = 1.114 g mL-1) instead of dextran sedimentation plus standard Ficoll-Hypaque, before discontinuous Percoll gradient centrifugation. Eosinophils from 11 controls and 11 eosinophilic patients were studied. The density distribution of eosinophils was similar in both groups. However, eosinophilic patients showed a significant increase in the absolute count of hypodense eosinophils. Normodense eosinophil purity and recovery were significantly higher in controls as compared with eosinophilic patients (p < 0.05). High-density Ficoll-Hypaque allowed separation of mononuclear and polymorphonuclear cells with high purity and recovery using a single centrifugation step. It also reduces one step in eosinophil purification as compared to the standard method. Thirdly, contrary to the single anti-CD16 magnetic bead separation method, it allows the isolation of normodense and hypodense eosinophils. In summary, this method is economic, reliable and permits the purification of different leucocyte populations.